The technical field of this invention relates to methods of decellularizing a tissue. In particular, decellularizing a tissue by chemically treating the isolated tissue with a series of oxidizing agents that remove the cellular and nuclear material from the isolated tissue while substantially retaining the biological and mechanical properties, as well as the biochemical composition of the resulting ECM.
Naturally occurring extracellular matrix (ECM)-based biomaterials are used for tissue repair and regeneration. One such extracellular matrix is small intestine submucosa (SIS). SIS has been used to repair, support, and stabilize a wide variety of anatomical defects and traumatic injuries. Commercially available SIS material is derived from porcine small intestinal submucosa that remodels to the qualities of its host when implanted in human soft tissues. Further, it is taught that the SIS material provides a natural matrix with a three-dimensional microstructure and biochemical composition that facilitates host cell proliferation and supports tissue remodeling. Indeed, SIS has been shown to contain biological molecules, such as growth factors and glycosaminoglycans that aid in the repair of soft tissue of the human body. The SIS material currently being used in the orthopedic field is provided in a dried and layered configuration in the form of a multilaminate patch. Several preclinical and clinical research studies have used laminated SIS to repair or regenerate soft tissue such as tendons (rotator cuff tendons, Achilles tendon, patellar tendon, flexor tendons etc.), ligaments (anterior and posterior cruciate ligaments, medial and lateral co-lateral ligaments), fibrocartilage (meniscus, labrum) and cartilage.
To date, some commercially produced ECM while useful for tissue regeneration, may still retain residual non-viable nucleic acids. These non-viable nucleic acids may leach into the surrounding environment during tissue regeneration. Although a number of different techniques have been employed to treat the ECM, few have successfully removed all of the residual nucleic acids without compromising mechanical, biological, and/or the the biochemical properties of the ECM.
Some studies have shown that remnants of degraded DNA are still present within the ECM after a cleaning and disinfecting process using a series of ethanol and peracetic acid washes. Some of the treatment methods also use chemicals which are difficult to wash out of the tissue (e.g., detergents), resulting in a high residual content of the chemical in the ECM, thus compromising its biocompatibility. Other methods using a combination of acids, bases and chelating agents, remove the proteoglycans and other non-collagenous proteins from the material (Janis, et al., (2003) Society for Biomaterials 29th Annual Meeting transactions, p. 272). Thus, these treatments remove the cellular remnants, but their effect on the mechanical properties of the material is usually detrimental.
U.S. Pat. No. 5,993,844 discloses a method for complete removal of nucleic acids from tissue to create an acellular matrix material that can be used as a biomaterial for tissue engineering or tissue repair purposes. However, the process disclosed in this patent calls for treatment of the ECM biomaterial with harsh bases like sodium hydroxide (NaOH), and/or acids like hydrogen chloride (HCl) in addition to treatment with chelating agents like EDTA. Such treatment leads to removal of nearly all nuclear material from the ECM, but at the same time also significantly removes non-collagenous material from the ECM biomaterial. While this may be desirable in some applications, other applications may require preservation of at least some of the non-collagenous components of tissue (e.g., growth factors, glycoproteins, glycosaminoglycans, etc.) to retain some of the biological properties of the material. Further, it has been documented that treatment with such harsh chemicals significantly compromises the mechanical properties of the ECM biomaterial (Janis et al., (2003) Supra), frequently requiring a chemical cross-linking step subsequent to the nuclear material removal step to strengthen the ECM biomaterial (Abraham et al., (2000) J Biomed Mater Res, 51:442-4552; Goleckner et al., (2000) J Biomed Mater Res. 52:365-373).
Therefore, there is a need for a treatment protocol that is effective in removing residual nucleic acids from an ECM without significantly altering the mechanical and biological properties, or the biochemical composition of the ECM biomaterial.